Expression Vector

ABSTRACT

The present invention provides an expression vector for cell-surface expression of proteins.

Since the discovery of monoclonal antibodies (mAbs) in 1975 by Kohler and Milstein, they have become molecular tools of inestimable value. Due to their high specificity, monoclonal antibodies (mAbs) are used for standard techniques throughout biology, being the key to the characterisation of protein function and distribution. Besides their usage in basic research, mAbs are also widely utilised as diagnostic and therapeutic agents. Due to this wide range of applications the generation of mAbs became a standard procedure. However, it can still be problematic, since for studies in physiological settings, it is important that the mAbs recognise the antigen in its native conformation.

Most commonly mAbs are raised against synthetic peptides derived from the predicted sequence of the target protein. Unfortunately, these Abs, though strongly reactive with peptide, frequently fail to recognise the native protein. Another standard procedure to generate mAbs uses recombinantly expressed protein. Prokaryotic expression systems are the most widely used expression hosts. But when studying mammalian surface proteins it is often necessary to use mammalian expression systems, as they are more likely to produce functional proteins with the appropriate disulfide-bonds, posttranslational glycosylations or proteolytic modifications. Purification of recombinant proteins is often a tedious undertaking, frequently representing a limiting step towards obtaining antibodies. Although introduction of affinity tags simplify purification, it often remains difficult to obtain recombinant protein in native conformation and in sufficient yield and purity. This applies most notably to membrane-associated proteins, as they are likely to lose their native structure during purification processes.

When attempting to generate mAbs capable of recognising the protein in its native context it is also critical to use protein in native conformation not only in the immunisation step but also for the screening procedure. Many standard hybridoma-screening protocols, such as the immobilisation of recombinant proteins on solid supports, may significantly alter protein conformation. For these reasons, mAbs selected on the basis of binding to a recombinant protein may not bind the same protein when it is in its native context.

Therefore, there is a need for an antigen expression system allowing the expression of antigens in native confirmation on the cell surface of cells.

In a first object the present invention provides a nucleic acid expression vector for cell-surface expression of proteins comprising in order a polynucleotide sequence encoding a secretion signal peptide, a cloning site for inserting a polynucleotide sequence encoding a protein to be expressed and a polynucleotide sequence encoding a transmembrane domain of glycophorin.

In a preferred embodiment of the nucleic acid expression vector, the transmembrane domain of glycophorin is the transmembrane domain of glycophorin A.

In a further preferred embodiment of the nucleic acid expression vector, the transmembrane domain of glycophorin A is the mouse glycophorin A transmembrane domain or the Armenian hamster glycophorin A domain.

In a further preferred embodiment of the nucleic acid expression vector, the mouse glycophorin A transmembrane domain comprises the amino acid sequence disclosed in Seq. Id. No. 1 and the Armenian hamster glycophorin A domain comprises the amino acid sequence disclosed in Seq. Id. No. 12.

In a further preferred embodiment of the nucleic acid expression vector, the secretion signal peptide is the secretion signal peptide of bee-venom melittin.

In a further preferred embodiment of the nucleic acid expression vector, the secretion signal peptide of bee-venom melittin comprises the amino acid sequence disclosed in Seq. Id. No. 2.

In a further preferred embodiment, the nucleic acid expression vector further comprises downstream (3′) of the cloning site for inserting a polynucleotide sequence encoding a protein to be expressed a polynucleotide sequence encoding a FLAG tag comprising the amino acid sequence of Seq. Id. No. 3.

In a further preferred embodiment, the nucleic acid expression vector further comprises downstream (3′) of the polynucleotide sequence encoding the transmembrane domain of glycophorin a polynucleotide sequence encoding a His tag, preferably a His tag comprising the amino acid sequence disclosed in Seq. Id. No. 4.

In a further preferred embodiment of the nucleic acid expression vector, the cloning site comprises the restriction enzyme cleavage sites of NheI, KpnI, BamHI, EcoRI, EcoRV and NotI.

In a further preferred embodiment, the nucleic acid expression vector comprises a polynucleotide sequence selected from the group consisting of Seq. Id. No. 5, Seq. Id. No. 13, Seq. Id. No. 14 and Seq. Id. No. 15.

In a further preferred embodiment of the nucleic acid expression vector, the protein to be expressed is a membrane associated protein.

a second object, the present invention provides a cell comprising the vector of the present invention, preferably a mammalian cell, more preferably a HEK cell.

In a third object, the present invention provides a method for the generation of monoclonal antibodies against a specific protein comprising the steps:

a) immunisation of a non-human animal with cells expressing on its cell surfaces the specific protein using the vector of the present invention,

b) isolating spleen cells of the non-human animals of step a),

c) fusing the spleen cells of step b) with myeloma cells to generate B cell hybridomas and

d) identification of B cell hybridomas expressing antibodies directed against the specific protein.

In a preferred embodiment of the method of the present invention, the non-human animal is a mouse or Armenian hamster.

“Nucleic acid expression vector” refers to an assembly which is capable of directing the expression of a sequence or gene of interest. The nucleic acid expression vector includes a promoter which is operably linked to the sequences or gene(s) of interest. Other control elements may be present as well. In addition, the vector may also include a bacterial origin of replication, one or more selectable markers, a signal which allows the vector to exist as single-stranded DNA (e.g., a M 13 origin of replication), a multiple cloning site, and a “mammalian” origin of replication (e.g., a SV40 or adenovirus origin of replication). A “vector” is capable of transferring gene sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (foreign) DNA may be generated.

The term “protein” as used herein, refers to a polymer of amino acids, and not to a specific length. Thus, peptides, oligopeptides and protein fragments are included within the definition of polypeptide.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein (1975) Nature 256:495, or may be made by recombinant DNA methods (see, e.g. U.S. Pat. No. 4,816,567 (Cabilly et al.) and Mage and Lamoyi (1987) in Monoclonal Antibody Production Techniques and Applications, pp. 79-97, Marcel Dekker, Inc., New York). The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al. (1990) Nature 348:552-554, for example.

SHORT DESCRIPTION OF FIGURES

FIG. 1A shows the primary structure of human ABCA1 (Seq. Id. No. 7), rat TMEM27 (Seq. Id. No. 9) and P. falciparum PFF0620c (Seq. Id. No. 11) proteins used in the examples. The domains used for the constructs described are marked with the diagonal lines with the amino acids at the N and C termini indicated;

FIG. 1B shows schematic diagrams of the expressed protein constructs derived from the vectors described in the examples. The extracellular domains are equivalent to the ones shown in FIG. 1A;

FIG. 2 shows a Westerm blot using anti-FLAG M2-HRP conjugated antibody (Sigma) of total cell lysates from HEK293 cells transfected with pANITA2-ABCA1 and pANITA2-TMEM27. Strong expression with bands at appropriate molecular weights is seen;

FIG. 3A shows cell-surface expression of PI-10620C on stably transfected HEK cells. Fluorescence (column 2 & 3) and differential interference contrast micrographs (column 1) of non-transfected HEK cells (line 1) and HEK cells displaying PFF0620C (line 2). Cells were grown on chamber-slides and stained without fixation with anti-FLAG antibody and FITC-labelled anti-mouse IgG antibodies. Nuclei were stained with DAPI;

FIG. 3B shows extracellular localisation of PFF0620C on stably transfected HEK cells. Fluorescence (line 1 & 3) and differential interference contrast micrograph (line 2 & 4) of PFF0620C-HEK cells after staining with anti-FLAG (left column) or anti-6× His antibodies (right column) and FITC-labelled anti-mouse IgG antibodies. With the anti-FLAG antibody living cells and methanol-fixed cells were stained, whereas the anti-His antibody only stained methanol-fixed cells, indicating intracellular localisation of the His-tag and extracellular localisation of the FLAG-tag together with the P. falciparum derived protein domain;

FIG. 4 shows the results of a screening of antibodies for binding to transfected cells. In a second step, all wells positive for IgG production were screened for antibody binding to transfected cells by IFA (immuno fluorescence assay). Transfected and non-transfected HEK cells spotted onto multiwell glass-slides were stained with individual hybridoma supernatants and analysed by fluorescence microscopy;

FIG. 5 shows a western blot analysis of the reactivity of generated monoclonal antibodies with the recombinant P. falciparum proteins. Specificity of representative monoclonal antibodies for the corresponding recombinant proteins is demonstrated by Western-blot analysis. Lysates of PFF0620c- (line 1), control pANITA2 constructs containing unrelated proteins (lines 2 & 3) and non-transfected HEK cells (line 4) were probed with anti-6× His mAb and an anti PFF0620cmAb generated as described, respectively.

FIG. 6 shows that PFD 1130w-specific monoclonal antibodies inhibit parasite growth in vivo.

EXAMPLES

Expression Proteins on the Cell Surface of Mammalian Cells.

The P. falciparum ORF PFF0620c, human ABCA1 extracellular domain and rat TMEM27 extracellular domain were expressed on the cell surface of HEK cells using the expression plasmids pANITA2-PFF0620C; pANITA2-ABCA1 or pANITA2-TMEM27 respectively. To ensure high levels of expression on the cell surface, the genes were modified in several ways (FIG. 1): i. the endogenous sequences were codon-optimised for expression in mammalian cells and only predicted extracelluar domains were used; ii. the endogenous secretion signal sequences were replaced by the secretion signal sequence of bee-venom melittin; iii. for membrane anchoring the transmembrane domain encoding sequence of mouse glycophorin A was used instead of the predicted GPI-attachment signal sequence or predicted transmembrane domains; iv. to allow expression analysis, a FLAG tag was inserted N-terminally of the transmembrane domain and a 6× His tag was placed at the C-terminus. The two tags were positioned just before and after the transmembrane domain to facilitate verification of the extracellular localisation of the recombinantly expressed antigens.

HEK-derived cell lines expressing P. falciparum PFF0620c, human ABCA1 extracellular domain and rat TMEM27 extracellular domain were established by stable transfection.

To obtain highly expressing cell lines, transfectants were separated into high-expressing cell-pools by fluorescent-activated-cell-sorting after surface staining with anti-FLAG antibodies. The mean fluorescence intensity of the cells gated for sorting into the high-expressing cell pool was 2.1-4.3 times higher than that of all transfectants.

Human ABCA1 and rat TMEM27-expressing cell lines were tested for expression by Western blot analysis, showing a high level of expression of a protein with the expected molecular weight. (FIG. 2) Cell surface expression of the P. falciparum PFF0620c protein was shown by immunofluoresence analysis with anti-FLAG antibody yielding strong signals on living cells. (FIG. 3) In contrast, staining with anti-6× His antibody gave strong signals only on methanol fixed cells but not on living cells (FIG. 3B). These results verified that PFF0620c is expressed and anchored in the cell wall with the FLAG-tag lying extracellularly and the His-tag lying intracellularly.

Development of malaria antigen specific antibodies in mice immunised with transfected HEK cells

The high-expressing cell pool of PFF0620c-HEK was used to immunise NMRI mice. Mice received intravenous injections of 10⁶ cells on three consecutive days and another suite of three daily injections two weeks later. Development of serum antibody titres was analysed by flow cytometry comparing immune-staining of the transfectant with that of non-transfected HEK cells. The fluorescence intensity observed with the transfectant was fourfold higher than that of non-transfected control HEK cells. This indicated that the mice had mounted an antibody response against the malaria antigen expressed on the surface of the transfected HEK cells.

Spleen cells of mice immunised with the transfected HEK cells were fused with PAI myeloma cells to generate B cell hybridoma. Fused cells were distributed in microtitre culture plate wells. To identify hybridoma cells that produce PFF0620c-specific antibodies a two-step screening procedure was used that completely obviates the requirement for purified recombinant proteins. First all culture wells were tested for IgG production by ELISA. Between 18 and 29%, of the tested wells were positive. In a second step all wells positive for IgG production were screened for antibody binding to transfected cells by IFA. Transfected and non-transfected HEK cells spotted onto multiwell glass-slides were stained with individual hybridoma supernatants and analysed by fluorescence microscopy (FIG. 4). Non-transfected HEK cells served as a negative control for each sample. Numerous clones positive on the transfected cells were also positive on non-transfected cells. However, the fusion yielded also numerous wells containing antibodies strongly reactive with the transfectant but not reactive with untransfected HEK cells. All other antibodies were specific for the transfected cells used for immunisation and did not stain control transfectants. From wells of this category, 17 hybridoma clones were derived by recloning from the PFF0620c-fusion.

The specificity of the monoclonal antibodies was further confirmed by Western blot analysis (FIG. 5). 16 of the mAbs stained the corresponding recombinant protein in the lysate of the transfectant used for immunisation, but not in lysates of control transfected or untransfected HEK cells.

PFD1130w-Specific Monoclonal Antibodies Inhibit Parasite Growth In Vivo

We evaluated the in vivo parasite inhibitory activity of anti-PFD1130w mAbs in a P. falciparum SCID mouse model. The anti-PFD1130w mAbs were produced using the same methods and vectors that were used for the generation of the mAbs against P. falciparum PFF0620c (see methods section below). This model uses non-myelodepleted NOD-scid IL2Rnull mice engrafted with human erythrocytes in order to allow the growth of P. falciparum. Groups of three mice with a parasitemia of 0.58±0.14% were injected once with 2.5 mg anti-PFD1130w c12 mAb, 0.5 mg anti-PFD 1130w c12 mAb or 2.5 mg isotype/subclass control mAb per mouse, respectively. Parasitemia of all mice was monitored for the next six days. While the parasitemia in mice that had received PBS only or the control mAb increased continuously, reaching 11.3±0.8% after six days, parasitemia of mice that received 0.5 mg anti-PFD1130w c12 mAb increased to a much lower extent, reaching 5.6±1.3% after six days. Parasitemia of mice receiving 2.5 mg anti-144)1130w c12 mAb stayed low till the end of the experiment (1.4±0.3% on day 6). The difference in parasitemia after 6 days compared to the negative control group was highly significant (two-sided t-test; P<0.0001) (FIG. 6).

The fact that anti-PFD1130w mAbs inhibit parasite growth in vivo indicates the power of the described entirely cell-based technology to generate mAbs that bind the endogenous protein in its native context.

Methods

Construction of Plasmids and Transformation

A double-stranded oligonucleotide encoding the secretion signal sequence of bee-venom melittin was ligated to NheI digested pcDNA3.1(+) (Invitrogen) resulting in plasmid pcDNA3.1_BVM, with a single NheI site retained 3′ of the signal sequence. A mouse glycophorin cytoplasmic and transmembrane domain cDNA was obtained by rtPCR (Invitrogen SuperScript III First Strand Synthesis kit and Roche Expand High Fidelity PCR System) using RNA extracted from bone marrow as a template. The resulting PCR amplicon being cloned into a pCR2.1 cloning vector. Primers to mouse glycophorin contained a 5′ NotI site and 3′ histidine tag followed by a stop codon and EagI site. The glycophorin-6His fragment was excised with EagI and ligated to NotI-digested pcDNA3.1_BVM resulting in plasmid pcDNA3.1_BVM_GP with the pcDNA3.1 NotI site preserved at the 5′ end of the glycophorin sequence. To create the finished expression vector (pANITA2) a double-stranded oligonucleotide was ligated into NotI-digested pcDNA3.1_BVM_GP encoding a Flag-tag flanked by short linker sequences and resulting in a unique NotI site to the 5′ side of the Flag-tag.

Rat TMEM27 extracellular domain (aa 15-130 of Seq. Id. No. 9); a predicted extracellular domain of P. falciparum gene PFF0620C (aa 21-353 of Seq. Id. No. 11) and human ABCA1 N-terminal extracellular domain (aa 43-640 of Seq. Id. No. 7) cDNA sequences were synthesised with optimisation of codon usage to give high expression in mammalian cell culture. The genes were ligated into the unique NheI and NotI sites of the pANITA2 vector and the sequence of the vectors confirmed by DNA sequencing. The resulting plasmids are hereafter referred to as pA-NITA2-TMEM27; pANITA2-PFF0620C or pANITA2-ABCA1 respectively.

In pANITA3.1 and pANITA3.3, the native pcDNA3.1 XbaI and XhoI sites were also removed by site-directed mutagenesis. The features of the multiple cloning sites and fusion-protein-coding sequences are shown in the table 1 below, with numbering from the insert start.

Armenian hamster glycophorin sequence was determined by PCR-cloning and nucleotide sequencing using the Chinese hamster glycophorin sequence as a guide for primer design and cDNA generated from Armenian hamster bone-marrow RNA preparations. The following sequences are depicted in table 1: pANITA2 with Kozak sequence=Seq. Id. No. 15, pANITA3.1=Seq. Id. No. 13 and pANITA3.3=Seq. Id. No. 14.

TABLE 1 Comparison of expression vectors Vector element pANITA2 pANITA3.1 pANITA3.3 Kozak sequence  1-12  1-12  1-12 Bee venom melittin signal  9-72  9-72  9-72 sequence Unique NheI restriction site 70-75 70-75 70-75 Unique KpnI restriction site 82-87 82-87 82-87 Unique BamHI restriction site 94-99 94-99 94-99 Unique EcoRI restriction site 106-111 106-111 106-111 Unique EcoRV restriction site 112-117 112-117 112-117 Unique XbaI restriction site — 118-123 118-123 Unique NotI restriction site 124-131 124-131 124-131 Flag tag/Enterokinase cleavage 133-156 133-156 133-156 site Unique HindIII restriction site — 154-159 154-159 Mouse glycophorin membrane 172-369 163-369 — anchor Armenian hamster glycophorin — — 178-375 membrane anchor 6-His tag 382-399 382-399 388-405 Stop codons 400-405 400-405 406-411

Establishment of HEK 293 Cell Lines Stably Expressing PFF0620C, TMEM27 or ABCA1 Domains.

293 HEK cells were transfected with pANITA2-TMEM27; pANITA2-PFF0620C or pA-NITA2-ABCA1 using JetPEI™ (PolyPlus) transfection reagent following the manufacturer's protocol. Antibiotic selection was started 48 h after transfection. The selection medium containing 500 ug/ml of Geneticin (Gibco) was exchanged every 3-4 days. After non-antibiotic resistant cells had died off and resistant cells started growing normally, a high-expressing pool was generated by FACS. Cells were dissociated with enzyme-free dissociation buffer (Cell dissociation buffer enzyme-free Hanks'-based, Gibco), washed with blocking buffer (PBS containing 3% BSA). The cells were then incubated with 200 μl of 100 μg/ml anti-FLAG mAb=FLAG-27 diluted in blocking buffer for 15 min on ice. The cells were then washed with blocking buffer and incubated with 200 μl of 100 μg/ml FITC-conjugated goat anti-mouse IgG antibodies (RAM/IgG(H+L)/FITC, Nordic Immunological Laboratories) diluted in blocking buffer for 15 min on ice. After a final wash the labelled cells were analysed and sorted using a BD FACSAria running FACSDiva software. All analyses were performed using appropriate scatter gates to exclude cellular debris and aggregates. Gating settings were set to collect highly labelled cells. Post-sorting, the cells were collected in culture medium with 20% FCS and plated in 35 mm wells

Immunofluorescence Staining of Living HEK Cells

For immunofluorescence staining of live HEK cells chamber slides (4-well chamber-slide, Lab-Tek™, Nunc™) were used. Wells were coated with 100 mg/l poly-D-lysine in H₂O in a humid box at room temperature over night. After washing the wells three times with sterile H₂O, 40′000 cells were seeded per well. Three days later the immunostaining was performed by incubating the wells with 500 μl of an appropriate mAb diluted in serum-free culture medium for 30 min on ice. After washing two times with serum-free culture medium 500 μl of 100 μg/ml FITC-conjugated goat anti-mouse IgG antibodies (RAM/IgG(H+L)/FITC, Nordic Immunological Laboratories) diluted in serum-free culture medium were added to the wells and incubated for 30 min on ice. Finally, the wells were rinsed twice with serum-free culture medium and once with DPBS (Dulbecco's Phosphate-Buffered Saline containing calcium, Gibco). The slides were mounted with mounting solution containing DAPI (ProLong® Gold antifade reagent with DAPI, Invitrogen) and covered with a coverslip. Stainings were assessed as described above.

Immunisation of Mice

NMRI mice were immunised by intravenous injections of 10⁶ stably transfected HEK cells. Cells were thawed, washed and resuspended in 0.9% NaCl. Injections were accomplished on three consecutive days and after two weeks again on three consecutive days. After the boost, blood was collected and the serum was tested for the presence of anti-PFF0620C antibodies by IFA using stably transfected 293 HEK cells.

Animals with serum strongly reactive with expressing cells were selected for fusion. These received a final injection of 10⁶ cells two and one day before the fusion. Mice were sacrificed and the spleen was removed. Spleen cells were harvested by trituration under sterile conditions and fused with the myeloma cell partner (PAI mouse myeloma cells, derived from P-3X63-Ag8) using polyethylene glycol 1500 (Roche Diagnostics). The fusion mix was plated into multiwell plates and hybridomas were selected by growing in HAT medium supplemented with culture supernatant of mouse macrophages P388. Wells were screened for specific IgG production between 2-3 weeks post-fusion by ELISA and IFA as described below. Cells from wells positive in initial screens were cloned by limiting dilution to obtain monoclonal populations.

IgG ELISA Screen

Maxisorp™ plates (Nunc) were coated overnight at 4° C. in a humid box with 100 μl of 5 μg/ml goat anti-mouse IgG (y-chain specific) mAb (Sigma) diluted in PBS. After two washings with PBS containing 0.05% Tween-20, wells were blocked with blocking buffer (50 mM Tris, 140 mM NaCl, 5 mM EDTA, 0.05% NONidet P40, 0.25% gelatine, 1% BSA) for 1 h at 37° C. and afterwards washed two times. 50 μl hybridoma supernatants were added to the wells and incubated for 1 h at 37° C. After washing 4 times, plates were incubated with 50 μl horseradish peroxidase-conjugated goat anti-mouse IgG (y-chain specific) (Sigma) diluted 1:1000 in blocking buffer for 1 h at room-temperature in a humid box in the dark. After washing 4 times, TMB peroxidase substrate solution was added and the colour change monitored.

Antibody Production and Characterisation

Identification of antibody isotypes was performed using a Mouse Monoclonal Antibody Isotyping Kit (ISO2, Sigma). For large-scale mAb production hybridoma cell lines were cultured in 500 ml roller-bottles (Corning). MAbs were purified by affinity chromatography using protein A or protein G Sepharose.

DNA and Protein Sequences

Gene/Protein name Species Description Seq. Id. No. Glycophorin A Mouse Transmembrane + cyto- 1 plasmic domain of glycophorin A Melittin Bee Secretion signal of bee 2 venom melittin Flag tag — Flag tag 3 His tag — His tag 4 Expression vector — Expression vector sequence 5 pANITA2 comprising secretion (without Kozak sequence) signal of bee venom melittin, cloning site for a protein to be expressed and transmembrane domain of mouse glycophorin A ABCA1 Human DNA encoding human 6 ABCA1 protein ABCA1 Human ABCA1 protein 7 TMEM27 Rat DNA encoding rat 8 TMEM27 TMEM27 Rat TMEM27 protein 9 PFF0620C Plasmodium falciparum DNA encoding 3D7 protein 10 PFF0620C Plasmodium falciparum 3D7 protein 11 Glycophorin A Armenian hamster Transmembrane + cyto- 12 plasmic domain of glycophorin A Expression vector — Expression vector sequence 13 pANITA3.1 comprising secretion signal of bee venom melittin, cloning site for a protein to be expressed and transmembrane domain of mouse glycophorin A Expression vector — Expression vector sequence 14 pANITA3.3 comprising secretion signal of bee venom melittin, cloning site for a protein to be expressed and transmembrane domain of Armenian hamster glycophorin A Expression vector — Expression vector sequence 15 pANITA2 with comprising secretion Kozak sequence (nt signal of bee venom 1-12) melittin, cloning site for a protein to be expressed and transmembrane domain of mouse glycophorin A

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference. 

1. A nucleic acid expression vector for cell-surface expression of proteins comprising in order a polynucleotide sequence comprising a sequence encoding a secretion signal peptide, a cloning site for inserting a polynucleotide sequence encoding a protein to be expressed and a polynucleotide sequence comprising a sequence encoding a transmembrane domain of glycophorin.
 2. The nucleic acid vector of claim 1, wherein the transmembrane domain of glycophorin is the transmembrane domain of glycophorin A.
 3. The nucleic acid vector of claim 1 or 2, wherein the transmembrane domain of glycophorin A is the mouse glycophorin A transmembrane domain or Armenian hamster glycophorin A domain.
 4. The nucleic acid vector of claim 3, wherein the mouse glycophorin A domain comprises the amino acids disclosed in Seq. Id. No. 1 and the Armenian hamster glycophorin A domain comprises the amino acid sequence disclosed in Seq. Id. No.
 12. 5. The nucleic acid vector of claims 1-4, wherein the secretion signal peptide is the secretion signal peptide of bee-venom melittin.
 6. The nucleic acid vector of claim 5, wherein the secretion signal peptide of bee-venom melittin comprises the amino acid sequence disclosed in Seq. Id. No.
 2. 7. The nucleic acid vector of claims 1-6, further comprising downstream (3′) of the cloning site for inserting a polynucleotide sequence encoding a protein to be expressed a polynucleoide sequence encoding a FLAG tag comprising the amino acid sequence of Seq. Id. No.
 3. 8. The nucleic acid vector of claims 1-7, further comprising downstream (3′) of the polynucleotide sequence encoding the transmembrane domain of glycophorin a polynucleotide sequence encoding a His tag, preferably a His tag comprising the amino acid sequence disclosed in Seq. Id. No.
 4. 9. The nucleic acid vector of claims 1-8, wherein the cloning site comprises the restriction enzyme cleavage sites of NheI, KpnI, BamHI, EcoRI, EcoRV and NotI.
 10. The nucleic acid vector of claims 1-9 comprising a polynucleotide sequence selected from the group consisting of Seq. Id. No. 5, Seq. Id. No. 13, Seq. Id. No. 14 or Seq. Id. No.
 15. 11. The nucleic acid vector of claims 1-10, wherein the protein to be expressed is a membrane associated protein.
 12. A cell comprising the vector of claims 1 to 11, preferably a mammalian cell, more preferably a HEK cell.
 13. Use of a cell of claim 12 for the expression of proteins suitable for antibody generation.
 14. Use of a cell of claim 12 for the immunisation of a non-human animal for antibody generation, preferably monoclonal antibodies.
 15. A method for the generation of monoclonal antibodies against a specific protein comprising the steps: a) immunisation of a non-human animal with cells expressing on its cell surfaces the specific protein using the vector of claims 1 to 11, b) isolating spleen cells of the non-human animals of step a), c) fusing the spleen cells of step b) with myeloma cells to generate B cell hybridomas and d) identification of B cell hybridomas expressing antibodies directed against the specific protein.
 16. The method of claim 15, wherein the non-human animal is a mouse or hamster.
 17. The invention as hereinbefore described. 